Disruption of cell cycle control mechanisms contributes significantly to the development of cancer in humans [Sherr, Cancer Res., 60:3689-95 (2000)]. Consistently, the two most frequently inactivated tumor suppressor genes in human cancer irrespective of tumor type, site, and patient age, are the p53 gene and the INK4a-Arf gene locus, both of which encode proteins involved in the regulation of cellular replication [Hall and Peters, Adv. Cancer Res., 68:67-108 (1996); Hainaut et al., Nucleic Acid Res., 25:151-157 (1997)]. The p53 gene encodes the transcription factor p53. Activation of the p53 gene in response to oncogenic stress signals results in cell cycle arrest or apoptosis, thereby enabling cells to repair genotoxic damage or alternatively, to be eliminated from the organism [reviewed in Ko et al., Genes & Devel. 10:1054-1072 (1996); Levine, Cell 88:323-331 (1997)]. Loss of p53 function cancels these surveillance functions thereby allowing defective cells to replicate and predisposing the cell to cancer development.
The INK4a/Arf gene locus has been shown to encode two unrelated proteins from alternative but partially overlapping reading frames: (i) p16INK4a and (ii) Arf (p14Arf in humans and p19ARF in the mouse) [Quelle et al., Cell, 83:993-1000 (1995)]. These proteins independently target two cell cycle control pathways. The N-terminal 62 amino acid residues of the 132 amino acid p14ARF protein and the N-terminal 63 amino acid residues of the 169 amino acid p19ARF protein are encoded by a unique first exon (1β), whereas the remaining amino acid residues are encoded by exon 2. An alternative reading frame of exon 2 also encodes the bulk of p16INK4a.
p16INK4a is an antagonist of cell replication. More specifically, p16INK4a inhibits the cyclin D-dependent kinases CDK4 and CDK6 [Serrano et al., Nature, 366:704-707 (1993)]. CDK4 and CDK6 play an important role in the cell replication cycle through their phosphorylation of the retinoblastoma protein (Rb). Hyperphosphorylation of Rb stimulates the cell to exit from the G1 phase and begin DNA synthesis, a required step prior to cell division. Thus, the inhibition of CDK4 and CDK6 by p16INK4a prevents hyperphosphorylated Rb-dependent DNA synthesis, thereby maintaining the cell in its non-replicating mode.
Disruption in mice of either the entire INK4a/Arf locus [Serrano et al., Cell, 85: 27-37 (1996)] or exon 1β [Kamijo et al., Cell, 91:649-59 (1997)] leads to multi-type tumor growth and early death, identifying Arf as a bona fide tumor suppressor. Interestingly, it has been suggested that disruption of INK4a does not contribute to spontaneous tumor formation in mice and that Arf disruption accounts for the high rate of spontaneous tumor formation in INK4a/Arf-null mice [Sherr, Cancer Res., 60: 3689-95 (2000)]. Since the INK4a/Arf locus is frequently disrupted in human cancers [Raus and Peters, Biochim. Biophys. Acta Rev. Cancer, 1378:F115-F177 (1998)], the loss of Arf function appears to be a major contributor to human cancers.
Indeed, Arf, in concert with other cell cycle regulators and tumor suppressors such as p53 and Rb, plays a central role in cellular responses to oncogenic stress, such as inappropriate mitogenic signaling. For example, Arf expression is activated by overexpression of proteins involved in mitogenic signaling, such as Myc [Zindy et al., Genes & Dev., 12:2424-2434 (1998)], E1A [de Stancbina et al., Genes Dev., 12:2434-42 (1998)], E2F [Bates et al., Nature, 395:124-5 (1998)], Ras [Palmero et al., Nature, 395:125-6 (1998)], and v-Ab1 [Radfar et al., Proc. Natl. Acad. Sci. U.S.A., 95:13194-13199 (1998)]. Activation of Arf leads to stabilization of p53 [Pomerantz et al., Cell, 92:713-23 (1998); Kamijo et al., Proc. Natl. Acad Sci., 95:8292-8297 (1998); Stott et al., Embo J, 17: 5001-14 (1998); and Zhang et al., Cell, 92:725-734 (1998)] followed by cell cycle arrest. Arf therefore connects the Rb and p53 pathways [Sherr, Cancer Res., 60: 3689-95 (2000)] so that excessive proliferative signaling via the Rb pathway activates arrest mechanisms controlled by p53.
Arf stabilizes p53 by interfering with an auto-regulatory loop involving p53 and Double Minute 2 (Hdm2 in humans, Mdm2 in mice) [Wu et al., Genes Dev., 7:1126-1132 (1993)] that maintains p53 at low levels under normal cellular conditions (i.e. in the absence of oncogenic stress, DNA damage, etc.). The positive component of this auto-regulatory loop involves activation of Mdm2 transcription by p53 [Barak et al., EMBO J, 12:461-468 (1993)]. The negative component has several facets. First, Mdm2 binds p53 [Kussie et al., Science, 274:948-953 (1996)] and inhibits the transactivation function of p53 [Oliner et al., Nature, 362:857-860 (1993); Momand et al., Cell, 69:1237-1245 (1992)]. Second, Mdm2 shuttles p53 from the nucleus to the cytoplasm and facilitates p53 degradation [Roth et al., Embo J, 17:554-64 (1998)]; Freedman et al., Mol Cell Biol, 18:7288-93 (1998)]. Third, Mdm2 acts as an E3 ubiquitin ligase toward p53 within the ubiquitin-dependent 26S proteosome pathway [Honda et al., FEBS Lett, 420:25-7 (1997)]. Therefore, Mdm2 inhibits p53 activity in the nucleus through multiple and diverse mechanisms. Balance between the positive and negative components of this auto-regulatory system is essential for cell survival. When p53 is inactivated, mice develop tumors at an unusually high rate [Donehower et al., Nature, 356:215-221 (1992)], indicating that p53-dependent tumor suppression is compromised. Additionally, when Mdm2 is inactivated, mice are not viable [Jones et al., Nature, 378:206-8 (1995); Montes de Oca Luna et al., Nature, 378:203-206 (1995)], suggesting that unregulated p53 expression is lethal. Mdm2−/− mice are rescued, however, by the additional inactivation of p53 [Jones et al., Nature, 378:206-8 (1995); Montes de Oca Luna et al., Nature, 378:203-206 (1995)]. Thus, proper regulation of p53 activity relies on appropriate balance between the positive and negative components of the p53-Mdm2 auto-regulatory system.
The first direct biochemical connection between p19ARF and p53 was established when it was found that p19ARF could bind to Mdm2, [Pomerantz et al., Cell, 92:713-723 (1998); Zhang et al., Cell, 92:725-734 (1998)]. Arf was subsequently found to inhibit the negative components of the p53-Mdm2 auto-regulatory loop by interfering with several of Mdm2's activities toward p53. First, by binding Mdm2, Arf inhibits Mdm2-dependent nucleo-cytoplasmic shuttling of p53 which leads to stabilization and activation of p53 [Tao et al., Proc Natl Acad Sci USA, 96:6937-41 (1999)]. Second, Arf inhibits the E3 ubiquitin ligase activity of Mdm2 toward p53 in vitro [Honda et al., Embo J, 18:22-7 (1999); Midgley et al., Oncogene, 19:2312-23 (2000)] and is thought to be an important aspect of Arf-dependent activation of p53 in vivo [Midgley et al., Oncogene, 19:2312-23 (2000); Llanos et al., Nat. Cell Biol., 3:445-452 (2001)]. Finally, Arf binds and sequesters Mdm2 in the nucleolus, physically separating Mdm2 and p53 in different sub-cellular compartments [Weber et al., Nat. Cell Biol., 1:20-26 (1999); Lohrum et al., Nat. Cell Biol., 2:179-81 (2000); Weber et al., Mol. Cell. Biol., 20:2517-2528 (2000)]. The relative importance of these three mechanisms to Arf-dependent stabilization and activation of p53 is a matter of debate. For example, a recent report shows that Mdm2 binding but not nucleolar localization is the functional property of Arf that is required for p53 activation [Llanos et al., Nat. Cell Biol., 3:445-452 (2001)]. This report, however, does not rule out earlier reports that nucleolar co-localization of Arf and Mdm2 contribute to p53 stabilization through sequestration [Weber et al., Nat. Cell Biol., 1:20-26 (1999)]. It is likely that Arf acts via several mechanisms to stabilize p53 and these have evolved in concert with the multiplicity of Mdm2's effects on p53. Importantly, direct interaction between Arf and Hdm2 is required for the multiple mechanisms of p53 stabilization.
Therefore, there is a need to further characterize the Arf-Hdm2 complex. In addition, there is a need to determine the specific domains of Arf and Hdm2 that are involved in this complex. Furthermore, there is a need to identify compounds that can mimic the effect of Arf on Hdm2, since the absence of functional Arf is commonplace in tumor cells. Alternatively, there is a need to identify compounds that inhibit the binding of Arf to Hdm2 to prevent undesired activation of p53-dependent pathways by, for example, DNA damaging agents, in normal cells.
The citation of any reference herein should not be deemed as an admission that such reference is available as prior art to the instant invention.